The budding yeast S. cerevisiae provides a surprising and remarkably useful model for longevity studies, because certain genetic and metabolic pathways regulating aging in yeast are evolutionarily conserved in metazoans, even in humans. In particular yeast provides a tractable genetic model for replicative aging, since yeast cells divide a certain number of times and then cease to replicate. A number of observations suggest that alterations in chromatin structure, including changes in histone covalent post-translational modifications, occur as yeast age. For example, experimental manipulation of Sir2 levels, a deacetylase that targets histones, changes the kinetics of aging - loss of Sir2 causes more rapid aging and over-expression of Sir2 causes delayed aging. However, whether chromatin changes are an important aspect of agingjs not well understood. Our hypothesis is that alterations of histone post-translational modifications and changes in chromatin structure cause decompaction of heterochromatic regions of the genome during yeast replicative aging and may directly contribute to the aging phenotype. We further hypothesize that the chromatin changes are epigenetic, in that they are a stable, inherited attribute of the aging yeast cell. In the proposed research we will test these hypotheses via several specific aims. (1) Our first goal is to determine whether Sir2 deacetylation of histone H416ac (a well-characterized substrate of Sir2) has a central physiological role to antagonize yeast aging. We will determine whether deacetylation of H4K16ac maintains compaction of heterochromatic regions in young yeast, and will investigate the mechanism of gradual decompaction as a key change in chromatin underlying aging. (2) Second, we will examine whether regulation of Sir2 is a critical aspect of aging. Sir2 function is reduced during yeast and metazoan aging, however the mechanistic basis of this has not been elucidated. We will determine whether the activity, regulation, and localization of Sir2 are altered, and if small molecule effectors can regulate Sir2 function during aging. (3) Finally, we will investigate a wider role of chromatin changes during aging including additional histone modifications, nucleosome remodeling, and altered histone composition. We will collaborate extensively with the other Projects within this Program. We will collaborate with Project 1 (R. Marmorstein) to examine physiological and small molecule activators and inhibitors of Sir2 during aging. We will collaborate with Project 3 (B. Johnson) to test conservation of Sir2 mechanisms in yeast replicative aging and replicative senescence in the telomerase deficiency model. We will collaborate with Project 4 (P. Adams) to investigate possible conservation in yeast of histone chaperone protein complexes relevant to heterochromatic foci that occur in the mammalian bone senescence model.